Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab9051 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

All lanes : Anti-Histone H4 (mono methyl K20) antibody - ChIP Grade (ab9051) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 peptide (ab14963) at 0.5 µg/ml
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (mono methyl K20) peptide (ab17043) at 0.5 µg/ml
Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H4 peptide (17-24) - di methyl K20 at 0.5 µg/ml
Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H4 (tri methyl K20) peptide (ab17567) at 0.5 µg/ml
Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml

Lysates/proteins at 0.5 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Observed band size: 13 kDa

why is the actual band size different from the predicted?


Exposure time: 10 seconds

IHC image of Histone H4 (mono methyl K20) staining in Human normal skin formalin fixed paraffin embedded tissue section*, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9051, 0.05µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Rabbit polyclonal to Histone H4 mono methyl K20 (1/250).

HeLa cells cultured on glass coverslips were fixed with 4% paraformaldehyde and then stained with ab9051 (green). Total chromatin was visualized using DAPI staining (red).

This image is part of the antibody characterisation at www.cellnucleus.com

Rabbit polyclonal to Histone H4 mono methyl K20 at 1/1000 on histone prep (Sigma).

Peptide competition (shown above lanes) at 1ug/ml.

This peptide blocking study shows that ab9051 is specific for Histone H4 (mono methyl K20). The activity of the antibody is specifically blocked by the addition of the H4 mono methyl peptide (ab17043).

Rabbit polyclonal to Histone H4 mono methyl K20 (1/250).

HeLa cells cultured on glass coverslips were fixed with 4% paraformaldehyde and then stained with ab9051 (green). Total chromatin was visualized using DAPI staining (red).

Left: merge; center: DAPI; right: Mono K20

This image is part of the antibody characterisation at www.cellnucleus.com

Rabbit polyclonal to Histone H4 mono methyl K20 (1/250).

Mouse 10T1 cells cultured on glass coverslips were fixed with 4% paraformaldehyde and then stained with ab9051.

Top left: metaphase cell. Mono methyl K20 (green); DAPI (red)
Top right: interphase cell. Mono methyl K20 (green); DAPI (red)
Lower left: interphase cell. Mono methyl K20 (green); DAPI (blue), TBP (red)
Lower right: interphase cell. Mono methyl K20 (green); DAPI (red)

This image is part of the antibody characterisation at www.cellnucleus.com

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